Expressed protein ligation, a novel method for studying protein-protein interactions in transcription

Expressed protein ligation is a novel protein semisynthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the ~600 amino acid σ⁷⁰ subunit of Escherichia coli RNA polymerase. The resulting semi- synthetic σ⁷⁰ constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567-600 of σ⁷⁰. The success of these semi- synthesis studies sets the stage for the future generation of semi-synthetic σ⁷⁰ molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.